The present disclosure relates to UNC-45A splice variants and their use in cancer therapeutics and diagnostics.
Approximately 90% of breast cancer deaths are caused by metastasis to bones, liver, lungs, or brain with a survival time for patients of 2 years. Cancer metastasis is tightly related to cell motility including cell invasion and migration in breast cancer.
UNC-45 functions as a molecular chaperone for myosin motors and as a co-chaperone for Hsp90 in both vertebrate and invertebrate animals. Myosins are actin-based motors that play critical roles in a variety of cellular processes, including cytokinesis, cellular trafficking, phagocytosis, maintenance of cell shape, and muscle contraction. Myosin-based movement results from a specific cycle of the myosin head binding and releasing ATP and actin. During this process, the myosin head goes through multiple folding conformations. Evidence from a variety of experimental systems indicates that myosins use specialized chaperones during their activity, folding, and assembly.
Molecular chaperones are necessary for de novo folding and structural maintenance of the myosin head. Expression of the myosin motor domain in bacteria results in misfolding. In vertebrate systems, the chaperonin containing TCP-1 (CCT), as well as molecular chaperones Hsp90 and Hsc70, are necessary but not sufficient in the folding of striated muscle myosin.
The UNC-45 family of molecular chaperones is necessary for the proper functions of myosins, the motor proteins of the actin cytoskeleton and the contractile thick filaments of the muscle and heart. In humans and other vertebrates, two genes have been discovered which encode UNC-45 chaperones. One encodes UNC-45A that is essential for embryonic development, cell migration, and cell division because of its role in the activation of both myosin IIA (MYH9) and Myosin IIB (MyH10). UNC-45A or its mouse ortholog UNC-45a is necessary for cell proliferation in mouse myoblasts and for cell migration and proliferation in metastatic human ovarian cancer cells.
Mutations in UNC-45/Cro1p/She4p(Dim1p) domain (UCS) proteins result in phenotypes related to defects in myosin folding and assembly. Reduced UCS domain protein function in fungal mutants produces myosins defective in actin:ATP transduction. In Caenorhabditis elegans, null unc-45 alleles results in embryonic arrest of body wall muscle development, and temperature-sensitive mutations lead to a paralyzed or uncoordinated phenotype at the restrictive temperature with marked disorganization of myofibrils. UNC-45 exerts chaperone activity in vitro on the myosin head and acts as a cochaperone that specifically binds Hsp90.
Mice and humans each have two genes that are located on different chromosomes, which encode distinct UNC-45-like protein isoforms, and are expressed either in multiple tissues or only in cardiac and skeletal muscles. Their expression is regulated during muscle differentiation in vitro, with the striated muscle isoform mRNA appearing during myoblast fusion.
UNC-45 is a substrate of an E3/E4-multiubiquitination complex containing CHN-1 (the C. elegans homologue of CHIP) and UFD-2. chn-1-null worms are viable and appear morphologically normal. However, UNC-45 overexpression leads to an uncoordinated phenotype in these worms, suggesting that increased levels of UNC-45 may cause muscle defects.
RNA interference (RNAi) pathway is often used in experimental biology to study the function of genes in a variety of in vitro and in vivo model systems. Double-stranded RNA is synthesized with a sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, researchers induce a drastic decrease in the expression of a targeted gene. Since RNAi may not totally eliminate the expression of the target gene, this technique is sometimes referred as a “knockdown”, to distinguish it from “knockout” procedures in which expression of a gene is entirely eliminated.